Problem: Small RNAs (including miRNAs, piRNAs, and endogenous siRNAs) are small, non-coding RNA molecules. Research shows that they have key roles in post-transcriptional gene regulation, such as cellular differentiation, cell death, and metabolic regulation. In general, an miRNA is composed of a highly conserved core sequence of 21–23 nucleotides within a less conserved precursor sequence, ranging in size from about 60 to over 120 nucleotides. Unfortunately, small RNAs are very difficult to clone.
Solution: Integrated DNA Technologies (IDT) has developed a new proprietary miRCat™ small RNA cloning system. The miRCat system permits cloning of very rare small RNAs and takes into account the natural variability in structure and sequence between species. Conveniently, it is compatible with most existing standard laboratory protocols for processes such as RNA extraction, purification, and cloning.
Based upon a pre-activated adenylated oligonucleotide linkering method, the miRCat cloning system has been carefully designed to make small RNA library creation easy for all researchers. It consists of three sequential protocols: RNA isolation and enrichment, followed by cloning linker attachment, and ending with amplification and cloning phases. RNA is isolated using an organic extraction method and is then isolated from the denaturing polyacrylamide gel using a synthetic 21-mer control (included in the kit). The purified small RNAs are 3’ ligated with a pre-activated, adenylated linker using T4 RNA ligase in the absence of ATP. Linkered RNAs are then purified from a second denaturing polyacrylamide gel and 5’ linkered with the 5’ M.R.S linker (included) using T4 RNA ligase in the presence of 10 nM ATP. These species are reverse transcribed and then PCR amplified using the primers included in the kit.
The system can be used for concatamer cloning or direct “shotgun” cloning; it also can provide PCR amplicons suitable for use with TOPO TA Cloning®* or pGEM® T-Easy** cloning vectors.
For cloning small RNAs lacking the 5’-phosphorylated end present in miRNAs, a modified kit — miRCat-33™ — has been developed, which uses 5’ ligationindependent cloning. This makes it an excellent cloning system for the recently discovered C. elegans 5’ tri-phosphorylated microRNAs, which would otherwise be omitted. Specific primers are also available to convert the resultant small RNA libraries using Roche/454 Life Sciences sequencing systems.
*TOPO TA Cloning is a registered trademark of Invitrogen Corporation
**pGEM® T-Easy is a registered trademark of Promega Corporation